Recent studies in Dictyostelium and human neutrophils challenge the prevailing view that autonomous cytoskeletal activity underlies cell motility and suggest instead that the chemotactic signal transduction network is a critical pacemaker of migration. The network is excitable and poised near threshold, accounting for its stochastic activation. Chemotactic gradients enhance excitability at the front and suppress it at the back. Adaptation to the average level of chemoattractant allows cells to sense gradients with a range of midpoint concentrations. This proposal is focused on the positive and delayed negative feedback loops required for excitability of the network as well as the mechanism of adaptation and directional sensing mediated at the level of the chemoattractant receptor and G?protein. A novel a sequestration knock?down strategy will be used to rapidly decrease Ras GTPases, PKBs, and PKB substrates, including several Rac GAPs, establish the role of the signal transduction network in motility, and delineate the links to the cytoskeleton. Chemoattractant?mediated spatiotemporal changes in activators, enzymes, and phosphoinositides will be determined and used to predict interactions. Preliminary findings in human neutrophils and mammalian cells will be pursued to generalize the concept of an excitable signal transduction network as a driver of migration. To delineate the feedback loops, cells will be clampedin a persistently spread morphology or induced to oscillate between spread and collapsed morphologies by genetic manipulation or synthetic lowering of PIP2 and the activation of network components will be quantified. The role of RasGefs N and S and PI5K in negative feedback will be assessed with the sequestration knock?down strategy. A tunable, light?controlled interacting protein strategy recently adapted to Dictyostelium, will be used to rapidly and reversibly activate Ras or decrease PIP2 in local regions of the membrane/cortex, bypass chemoattractant receptors, and directly elicit pseudopods to guide cells. An in vitro planar bilayer assay and single molecule imaging will be used to investigate by the properties that control binding of PI3K and PTEN and other signal transduction components to the membrane. Chemoattractants reversibly suppress network activity cells lacking G?, providing a powerful tool to investigate adaptation. Using this, it will be confirmed that receptor phosphorylation is not the adaptation mechanism. Then proteins that are phosphorylated or undergo modifications consistent with the properties of adaptation will be identified and their rol in the process determined. Considering the key role of this fascinating process in cell physiology, our systems approach to understanding chemotaxis promises to eventually lead to new therapeutic strategies for a host of diseases.